Bacteria and archaea protect themselves from attack by foreign genetic elements such as plasmids, transposons, and phages, using the CRISPR-Cas adaptive immune system.1, 2, 3 CRISPR-Cas systems are incredibly diverse and widespread in nature, with approximately 40% of bacteria and 90% of archaea encoding one or more systems.4, 5 They are broadly classified into two classes and six types.6 Class 1 systems (types I, III, and IV) have multi-protein surveillance complexes, while Class 2 systems (types II, V, and VI) use single protein effector molecules. The type II systems, which use Cas9 effector proteins, are the most intensively studied and widely used for genome editing purposes.7

Type II CRISPR-Cas systems are subdivided into subtypes (II-A, II-B, and II-C) based on the genetic organization of CRISPR loci and sequence similarity of the Cas9 proteins.6 The type II-A system from Streptococcus pyogenes (SpyCas9), is currently widely used in genome editing,7 and type II-C Cas9 systems like Neisseria meningitidis (NmeCas9) are being explored as more compact and accurate alternatives to SpyCas9.8 Type II CRISPR-Cas immunity is mediated through the activity of the Cas9 protein and two small RNAs known as the CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). Complementary segments of the tracrRNA and the crRNA interact to form the guide RNA. The crRNA and tracrRNA are commonly engineered into a single guide RNA (sgRNA) for Cas9-mediated genome editing applications.

The biological activity of CRISPR-Cas9 can be divided into three stages: (1) guide RNA loading, (2) recognizing and binding to the guide RNA-targeted foreign DNA sequence, and (3) cleaving the target DNA.7, 9, 10 The multiple domains required for these processes are contained within two regions of Cas9 known as the recognition (REC) lobe and the nuclease (NUC) lobe (Figure 1(a)). The REC lobe, which is important for guide RNA recognition, is composed of two or three REC subdomains and an arginine-rich bridge-helix (BH) that links to the NUC lobe and is crucial for guide RNA-DNA recognition. The NUC lobe includes the HNH and RuvC endonuclease domains, which cleave the foreign DNA target and nontarget strands, respectively. Two linker regions (Linker 1 and 2) flank the HNH domain and allow it to move and access the target DNA molecule. The NUC lobe also contains the wedge (WED) domain and the PAM-interacting domain (PID) which surveys the incoming foreign DNA, binds the protospacer adjacent motif (PAM) located immediately upstream of the DNA target, and allows subsequent unwinding of the target DNA in preparation for pairing with the guide RNA.7 Following this pairing, the HNH and RuvC endonuclease domains cleave the foreign target DNA.

Type II CRISPR-Cas9 proteins typically range in size from ∼800 to ∼1400 amino acids.11 Type II-B Cas9s are generally the largest, with the Francisella novicida Cas9 (FnoCas9) at 1629 residues12 and type II-C Cas9s are generally the most compact, with Neisseria meningitidis Cas9 (Nme1Cas9) and Campylobacter jejuni Cas9 (CjeCas9) consisting of only 1083 and 984 residues, respectively.13, 14 The size differences are primarily due to variable REC lobes; the REC lobes of larger Cas9s are composed of three REC domains, while smaller Cas9s, such as SauCas9 and the type II-C Cas9s, have only two REC domains (Figure 1(b)).

To circumvent bacterial CRISPR-Cas defence, phages encode small proteins that inactivate CRISPR-Cas systems. In 2013, the first of these protein inhibitors, known as “anti-CRISPR” proteins, were discovered against the type I-F CRISPR-Cas system15 and in 2016 the first anti-CRISPRs that inactivate type II CRISPR-Cas9 systems were described.16 Anti-CRISPR proteins are named after the type of CRISPR-Cas system being inhibited, followed by a number according to the order of discovery. For example, the first anti-CRISPR discovered to inhibit the type II-C CRISPR-Cas system was designated AcrIIC1.16 Numerous reports of additional type II anti-CRISPR proteins soon followed and an online database (https://tinyurl.com/anti-CRISPR) was created to register the names of newly discovered anti-CRISPR proteins.[18] There are currently 38 distinct families of proteins described that inhibit a variety of CRISPR-Cas9 systems. Studies of these type II anti-CRISPR proteins have shown that phages incorporate a tremendously diverse arsenal of proteins to combat Cas9-based immunity. In this review, we explore the various inhibitory mechanisms of all reported type II anti-CRISPR proteins.